ABSTRACT
Objective To investigate the expression of miR-650 in osteosarcoma tissue and cell lines and its action mechanism in the osteosarcoma formation.Methods The realtime fluorescence quantitative PCR was used to detect and compare the expres-sions of miR-650 between osteosarcoma tissue and normal tissue or between osteosarcoma cell lines(MG63)and normal human os-teoblast cell lines(hFOB1.19);the MTT experiment was used to investigate the proliferation situation in different groups;Western blot was used to detect the ING4 protein expression.Results The expression of miR-650 in osteosarcoma tissue and MG63 cells was higher than that in normal tissue and human osteoblast cell;after inhibiting miR-650 expression,the proliferation ability of MG63 was significantly decreased.Moreover the expression of inhibitor of growth 4(ING4)was significantly increased when miR-650 was reduced,ING4 mRNA of negative control group,scramble group and miRNA group were 1.00 ± 0.16,1.08 ± 0.14 and 5.35 ± 0.32 respectively;the ING4 protein expressions were 0.62 ± 0.06,0.59 ± 0.12 and 2.45 ± 0.20 respectively;compared with negative control group,the difference was statistically significant(P<0.05);but after suppressing this elevation,the proliferation a-bility of MG63 cells had a certain recovery.Conclusion MiR-650 promotes MG63 proliferation via reducing ING4 expression,which suggests that miR-650 could be a new target of treating osteosarcoma.
ABSTRACT
Objective To investigate the effects of inhibitor of growth 4(ING4)on the proliferation,migration and the expression of angiogenesis related factors such as VEGF,MMP-2,MMP-9 in endothelial cells. Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro;ING4 plasmid and siRNA were constructed and transfected to HUVECs;the proliferation of HUVECs was evaluated by MTT assay;the ability of migration was evaluated by Transwell assay;real-time PCR and Western blotting were used to determine the expression of mRNA and pro-tein of angiogenesis related factors such as VEGF,MMP-2,and MMP-9. Results MTT and Transwell assay showed that ING4 has the ability to inhibit the proliferation and migration of HUVECs,and the results of real-time PCR and Western blotting proved that ING4 can inhibit the expres-sion of angiogenesis related factors such as VEGF,MMP-2,and MMP-9. Conclusion ING4 can inhibit the proliferation and migration of HU-VECs,down-regulate the expression of angiogenesis related factors such as VEGF,MMP-2,MMP-9,and inhibit angiogenesis.
ABSTRACT
Objective To construct a recombinant adenoviral vector carrying and co-expressing human inhibitor of growth 4(ING4) and human interleukin-24(IL-24) mediated by poly( A)-Promoter[Ad-ING4-poly(A)-Promoter-IL-24, referred to as Ad-ING4-IL-24] and explore its effect on the growth of HepG-2 human hepatocellular carcinoma cellsin vitro. Methods The poly(A)-Promoter(hEFl-elF4g) (Sal Ⅰ and Not Ⅰ ), ING4 ( Bgl Ⅱ and Sal Ⅰ ), and IL-24 ( Xho Ⅰ and Xba Ⅰ ) fragments were amplified by PCRusing pORF-mbcl-2α, pcDNA3.0-IL-24, and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-poly (A)-Promoter-lL-24, respectively. The pAdTrack-CMV-ING4-poly (A)-Promoter-IL-24 transfer vector linearized with Pme Ⅰ digestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-l-pAdTrack-CMV-ING4-poly ( A )-Promoter-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰ digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by liposome, leading to formation of the recombinant adenoviruses Ad-ING4-IL-24co-expressing ING4 and IL-24. The Ad-ING4-IL-24 were amplified in QBI-293A cells and its titer was up to 3.5 × 109 PFU/ml. Adenovirus-mediated ING4 and IL-24 genes expression in HepG-2 cells was examined by RT-PCR and Western blot. The growth-suppressing and apoptosis-inducingg effect of Ad-ING4-IL-24 coexpressing ING4 and IL-24 on HepG-2 human hepatocellular carcinoma cells was assessed by MTT assay and FCM, respectively. Results DNA sequencing showed that the ING4, poly (A)-Promoter, and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in HepG-2 cells. Adenovirusmediated ING4 and IL-24 co-expression significantly suppressed HepG-2 hepatocellular carcinoma cell growth and induced cell apoptosis, and the effect of Ad-ING4-IL-24 group was more significant than AdING4 and Ad-IL-24 group. Conclusion The adenoviral vector co-expressing ING4 and IL-24 mediated by poly(A)-Promoter(Ad-ING4-IL-24) was successfully constructed. Ad-ING4-IL-24 had marked anti-tumor effect in suppressing HepG-2 human hepatocellular carcinoma cell growth and inducing cell apoptosis in vitro. Compared with Ad-ING4 and Ad- IL-24, Ad-ING4-IL-24 enhanced anti-tumor effect.